Contraction of the heart occurs through regulated interactions of the myofilament proteins in response to increasing intracellular calcium concentrations. Recent studies have shown that specific post-translational modifications to the myofilament regulatory protein, troponin I are involved in the progression and development of ischemia/reperfusion injury. Furthermore, alterations in the phosphorylation of troponin I are associated with heart failure. The underlying hypothesis of this proposal is that disease related posttranslational modifications of cardiac troponin I produce abnormalities of cardiac function that may be delineated by creating and characterizing in vivo models. The long-range goal of this work is to understand the underlying molecular mechanism by which these variants alter cardiac function in order to design strategies to prevent or treat cardiac dysfunction. The dissection of the molecular pathophysiology of troponin I post-translational modifications will be approached in a series of highly collaborative integrative studies focused on modeling of myofilament disease-related proteomic changes in vivo. We will also exploit a transgenic model of troponin I proteolysis we have developed in order to dissect other potential modifiers in the progressive cardiomyopathy in these mice. To address these goals the following aims are proposed: 1. To assess genomic and proteomic changes associated with the developmental progression in the phenotype of dilated cardiomyopathy in the troponin I 1-193 mice, with the goal of identifying early alterations in the genome or specific subproteomes. 2. A. To determine whether constitutive "pseudo" phosphorylation of troponin I at protein kinase A sites protects the heart from deleterious effects of heart failure including a diminished force frequency response and delayed relaxation in response to increased afterload. B. To determine whether alterations in site-specific phosphorylation of troponin I by protein kinase A and protein kinase C associated with heart failure have a primary deleterious effect on cardiac muscle function in vitro and myocardial function in vivo. C. To determine the effect of a novel phosphorylation of troponin I at Serine 150 by p21activated kinase in vivo. This work should provide insight into the in vivo effects of specific post-translational modifications of the myofilaments, which contribute to pathophysiology of ischemic myocardial diseases and heart failure. In the long-term this will contribute to the development of novel therapies. [unreadable] [unreadable] [unreadable]